Pulmonary endothelium is the locus of early structural and functional changes in hyperoxic lung injury. An imbalance between the production of partially reduced oxygen species and their elimination appears to account for the genesis and/or maintenance of such pathology and evidence exists that such injury may be exacerbated by partially reduced nitrogen species. Nonetheless, it is apparent that nitric oxide (NO) may actually limit endothelial cell injury. The molecular mechanism underlying the protective role of NO, especially in pulmonary endothelium, remains unknown. From recent reports and preliminary data, we hypothesize that iNOS derived NO may be protective to lung endothelium by its potential antiapoptotic effects mediated by posttranslational S-nitrosation of proteins. Furthermore, we have recently shown that S-nitrosation of zinc thiolate clusters in metallothionein (MT) is a critical component of cellular redox sensitivity linking NO to zinc homeostasis in pulmonary endothelial cells. The well known contributions of zinc to transcriptional activity and the inhibitory effects of zinc on apoptosis underscore the importance of SNO-MT in the signaling pathway underlying NO mediated cytoprotection. Accordingly we will determine the: Aim I: role of iNOS derived NO in affecting pulmonary endothelial cell structure and function in hyperoxia. We hypothesize that iNOS derived NO is cytoprotective in lung. iNOS-/- mice, endothelial cell transgenic mice and anti-PECAM targeted somatic gene transfer of iNOS to pulmonary endothelium will be used to test this hypothesis in pulmonary circulation of intact mice exposed to hyperoxia. Aim II: mechanism by which NO is antiapoptotic in cultured murine lung endothelial cells (MLEC). We hypothesize that NO mediated release of zinc underlies antiapoptotic effects of iNOS derived NO in LPS induced apoptosis in MLEC. Full spectral confocal and multiphoton laser scanning microscopy (MPSLM) of GFP-labeled metal regulatory factor-1 and fluorescence resonance energy transfer (FRET) will be done. Aim III: role of zinc in NO induced protection of hyperoxic injury to pulmonary endothelium in intact mouse lung. We hypothesize that release of zinc is an important contributing factor to NO mediated resistance to hyperoxia in intact mice. NO induced changes in labile zinc via MPSLM and FRET in perfused lung and zinc depletion or targeted disruption of MT on sensitivity of intact mice to hyperoxia will be studied.